I. Field of the Invention
The present invention relates generally to the fields of protein chemistry and developmental biology. More particularly, it concerns peptide segments from the extracellular domain of syndecan-1 (Sdc-1) that can inhibit angiogenesis and can thus be used to treat angiogenesis in pathologic conditions.
II. Description of Related Art
A. Function of αvβ3 and αvβ5 Integrins in Angiogenesis
Several different growth factors, among them fibroblast growth factor (FGF) and vascular endothelial cell growth factor (VEGF), are often released by tumors to cause endothelial cells to undergo angiogenesis. Blood vessels in the vicinity of the tumor respond to VEGF by becoming leaky (thus the alternate name “vascular permeability factor”) allowing fibronectin, vitronectin and fibrinogen in the blood to infiltrate the surrounding matrix. These matrix ligands are critical adhesion and activation ligands for the αvβ3 and αvβ5 integrins, which have roles in the chemotactic migration of the endothelial cells and in the survival of the cells during vessel pruning. A second response to the growth factors, particularly FGF, is the activation of a neovessel development program that relies on Hox master genes (Boudreau et al., 1997; Myers et al., 2000; Myers et al., 2002). HoxD3 is initially expressed and controls a family of genes that are necessary for the initial migration process, including upregulation of the αvβ3 integrin, MMPs and uPAR (Boudreau et al., 1997). Expression of HoxD3 is followed by HoxB3, which regulates the morphogenesis leading to the formation of small vessels (Myers et al., 2000), and finally by the HoxD10 gene, which restores the mature phenotype of the cells (Myers et al., 2002); it is HoxD10 that is expressed in resting, stable blood vessels in vivo.
The αvβ3 and αvβ5 integrins are important not only during endothelial cell migration, but are important players in the survival of the endothelial cells. Although endothelial cells in mature vessels are not readily susceptible to apoptosis, angiogenic cells that are induced by growth factors are highly susceptible and rely upon the continued presence of these factors for survival. This is shown experimentally by inducing angiogenesis with VEGF and causing apoptosis by its withdrawal, and is demonstrated in vivo when the developing ovarian follicle induces angiogenesis by release of VEGF and the newly formed bloods vessels regress when the source of VEGF is lost upon ovulation. Endothelial cells responding to VEGF rely on signaling from the αvβ5 integrin in order to prevent this apoptotic process (Brooks et al., 1994; Friedlander et al., 1995). Similarly, endothelial cells responding to fibroblast growth factor (FGF) are susceptible to apoptosis unless there is coordinate signaling from the αvβ3 integrin. Thus, inhibitors that disrupt the activation of these two integrins are potential drugs for blocking angiogenesis not only because they can prevent the positive signaling from the integrins that aid in endothelial cell migration and formation of new vessels, but also because they have the potential to elicit “negative” signals that trigger apoptosis and death of the endothelial cell. Furthermore, this mechanism is not confined to endothelial cells and would apply as well to tumor cells or other cells that rely on either of these integrins to initiate disease processes.
B. Syndecans
The syndecan family of cell surface heparan sulfate (HS) proteoglycans is comprised of four vertebrate members. These receptors are expressed on virtually all cell types, although their expression may be altered in disease states such as cancer (Beauvais and Rapraeger, 2004). The syndecan core proteins share a high degree of conservation in their short cytoplasmic and transmembrane (TM) domains; in contrast, their ectodomains (EDs) are divergent with the exception of attachment sites for HS glycosaminoglycans. Via their HS chains, syndecans regulate the signaling of growth factors, chemokines, and morphogens and engage components of the ECM including VN, FN, LN, tenascin, thrombospondin, and the fibrillar COLs (Bernfield et al., 1999).
In addition to the activities of their HS chains, the syndecan core proteins have roles in cell adhesion signaling (Rapraeger, 2000; Tumova et al., 2000). Conserved and variable regions of the syndecan cytoplasmic domains appear critical for binding interactions that lead to adhesion-mediated signaling and reorganization of the actin cytoskeleton (Couchman et al., 2001). Important roles for the TM domain have also been demonstrated for Sdc-1 and syndecan-4 (Sdc-4) (Tkachenko and Simons, 2002; McQuade and Rapraeger, 2003). Perhaps the least expected active protein domain is the syndecan ED, which bears the HS chains. Nonetheless, several emerging studies suggest that the syndecan ED may have important regulatory roles in cell adhesion signaling. Cell spreading and morphogenetic activities in COS-7 and Schwann cells trace in part to the S1ED (Carey et al., 1994; Adams et al., 2001). Raji cells require the Sdc-1 TM domain for initial spreading, but depend on a S1ED activity for cell polarization (McQuade and Rapraeger, 2003). Moreover, inhibition of ARH-77 myeloma and hepatocellular carcinoma cell invasion into a COL I matrix by Sdc-1 also traces to a region of its extracellular core protein domain (Liu et al., 1998; Ohtake et al., 1999).
The activities of other syndecans also trace to their EDs. Overexpression of Syndecan-2 (Sdc-2) in COS-1 and Swiss 3T3 cells induces filipodial extension and deletion mutants of Sdc-2 map activity to the S2ED (Granes et al., 1999). Upregulation of Sdc-2 expression in colon carcinoma cells leads to altered cell morphology and colony formation in soft agar; treatment with recombinant S2ED disrupts these behaviors (Park et al., 2002; Kim et al., 2003). Finally, activated B-lymphocytes, when seeded on S4ED antibodies, exhibit morphological changes and filipodial extensions. Intriguingly, only the S4ED is required for this response, indicating that it may interact with a TM partner to transmit a dendritic signal (Yamashita et al., 1999).
C. αvβ3 Integrins are Regulated by Syndecan-1
The inventors' previous work in the MDA-MB-231 cells suggested that cell spreading induced upon anchorage of the cells to a Sdc-1 antibody relies on functional coupling of the syndecan to activated αvβ3 integrins (Beauvais and Rapraeger, 2003). This spreading response is rapid (˜15-30 min) and occurs even in the absence of an integrin ligand (i.e., spreading is not blocked by cycloheximide or EGTA treatment), so long as the cells are adherent via Sdc-1. Intriguingly, the αvβ3-dependent spreading mechanism is blocked by the addition of soluble, recombinant Sdc-1ED, suggesting that anchorage of Sdc-1 to a ligand provides a platform for αvβ3 integrin activation and adhesion signaling via binding interaction of the syndecan ED. These findings raised a fundamental question about the role of Sdc-1 in ECM signaling, in particular whether or not Sdc-1 is required for αvβ3 activation and signaling in response to a native matrix ligand. The inventors went on, in subsequent studies to show that Sdc-1 is required for signaling though both αvβ3 and αvβ5, and that inhibition of this function by competitive binding (Sdc-1ED) or siRNA inhibition blocks cell attachment, cell spreading, cell migration and angiogensis (unpublished).